PROJECT SUMMARY Cell surface glycans mediate interactions with receptors on other cells, in the extracellular matrix, or on the same cell membrane. Altered glycosylation has long been known as a hallmark of cancer. Two frequently observed cancer-associated phenotypes are hypersialylation and mucin overexpression. These cancer glycosignatures strongly correlate with disease aggressiveness and poor patient outcomes, but their functional contribution to cancer progression has been unclear. The broad objective of this program is to bring chemical tools to bear on this important problem in oncology, with an eye for developing new modes of intervention. An enabling tool for these studies are synthetic glycopolymers that we used to engineer discrete glycosylation patterns on live cells, or to engage specific glycan-binding proteins in a multivalent manner. In the previous granting period we made three major discoveries regarding the roles of cancer glycosignatures in disease: (1) Hypersialylation is a mechanism of immune evasion mediated through the Siglec family of sialic acid-binding immune cell receptors. Accordingly, immune cell killing of cancer cells can be potentiated by targeted cleavage of their cell-surface sialosides using antibody-sialidase conjugates. (2) Mucin overexpression enhances the thickness and stiffness of the glycocalyx, which promotes integrin clustering and focal adhesion signaling. This, in turn, enhances cell survival in vitro and promotes metastasis in mouse tumor models. And finally, (3) a glycan switching mechanism modulates partitioning of galectin-1, a prominent breast cancer marker, between a cell's glycocalyx and nucleus. Nuclear localization of galectin-1 drives breast cancer invasion, and this is inhibited by glycopolymers that sequester galectin-1 extracellularly. These discoveries form the foundation of the aims proposed in this renewal application. Aim 1 is a corollary to our discovery that cancer mucins drive oncogenesis. We will develop antibody-enzyme conjugates comprising mucin-specific proteases (aka ?mucinases?) to deforest cancer cells. We will generate tool molecules using known bacterial mucinases, and also identify human mucinases for incorporation into therapeutic candidates. In Aim 2, we will construct next-generation glycopolymers with native polypeptide backbones. These will be employed for fundamental studies of cancer glycobiology and for translational applications in Aim 3. Finally, in Aim 3 we introduce a new strategy for targeting extracellular proteins for degradation using glycopolymers that hijack the mannose-6-phosphate receptor (M6PR) lysosomal trafficking pathway. We will construct antibody-M6P glycopolymer conjugates that bind oncogenic cell-surface molecules such as growth factor receptors and the cancer-associated mucin MUC1 and target them for lysosomal degradation via engagement of M6PR. This new therapeutic modality complements the popular PROTAC approach for targeting intracellular proteins for proteasomal degradation.